Complex of Hepatitis B virus polymerase with p11 and method for controlling movement of the complex in HepG2 cell

ABSTRACT

Disclosed is a complex of the 11 kDa Ca 2+  binding protein of 11 and Hepatitis B virus polymerase (HBVPol) which moves to Promyelocytic Leukemia Nuclear Body (PMLNB). Furthermore, the present invention discloses a method for controlling movement of the HBVPol/p11 complex into the nucleus of HepG2 cell with adjusting of intracellular Ca 2+  ion concentration by administrating an agent for controlling calcium ion concentration in HepG2 cells.

FIELD OF THE INVENTION

[0001] The present invention relates to a complex of the 11 kDa Ca²⁺binding protein and Hepatitis B virus polymerase (HBVPol) which isinvolved in Hepatitis B.

BACKGROUND OF THE INVENTION

[0002] A connection with liver cancer with infection by Hepatitis Bvirus (HBV) and use of interferone or a nucleoside analogue for treatingthe HBV infection, have been reported. However, such treatment has beenapplicable only for some of the infected patients and could not removeHBV completely. Therefore, there is a need for substrate materials toenhance the curative efficiency against HBV infection.

[0003] Studies for finding intracellular factors which bind to HBVPolhave been well advanced, and Pol of duck hepatitis B virus, which bindsto Hsp90, p23 and Hsp70, shows a possible important role in the virusgrowth (Hu, J. et al., Hepadnavirus assembly and reverse transcriptionrequire a multi-component chaperone complex which is incorporated intonucleocapsids, EMBO J., 16 (1997) 59-68). Although this research was notcarried out on HBV capable of infecting humans, HBVPol with multipleenzymatic activities as a single protein, has high possibility ofbinding intracellular factors and is believed to complete the multipleenzymatic activities through such processing.

[0004] HBVPol is expressed in a very low level in E. coli or yeast sothat biochemical and structural research therefor are not feasible. Forthis reason, the live viruses have been used or experiments through invitro eukaryotic expression have been performed. However, theseexperiments pose a danger according to how they are conducted and theconsequent performance thereof involves high costs required to carrythem out safely.

SUMMARY OF THE INVENTION

[0005] The object of this invention is to provide a carcinogenicmechanism for HBV and provide information for treating cancer by cloninga protein capable of binding to Hepatitis B virus polymerase (HBVPol)and identifying the location of both HBVPol and the protein in humanliver to understand the correlation with cell organelles in which theyare present.

[0006] Specifically, the present invention provides a complex of the 11kDa Ca²⁺ binding protein and HBVPol, which is involved in Hepatitis B.The structure and amino acid sequence for the 11 kDa Ca²⁺ bindingprotein is published in Nat Struct Biol. 1999 Jan; 6(1):89-95 and theamino acid sequence for the Hepatitis B virus polymerase is published inToh, H., Hayashida, H. and Miyata, T. 1983. Sequency Homology BetweenRetroviral Reverse Transcriptase and Putative Polymerases of Hepatitis BVirus and Cauliflower Mosaic Virus. Nature; 305: 827-829.

[0007] A further object of the invention is to provide a method forcontrolling movement of the complex in the HepG2 cell.

[0008] The invention comprises a complex of the 11 kDa Ca²⁺ bindingprotein p11 and Hepatitis B virus polymerase, referred to herein as theHBVPol/p11 complex. This complex is characterized as moving to thePromyelocytic Leukemia Nuclear Body (PMLNB). The invention alsocomprises a method for controlling movement of the HBVPol/p11 complexinto the nucleus of a HepG2 cell through adjustment of intracellularconcentration of Ca²⁺ ion by administrating an agent which controls thecalcium ion concentration in the HepG2 cell. Agents which controlcalcium ion concentration can increase or decrease the intracellularconcentration of Ca²⁺ ion.

BRIEF DESCRIPTION OF DRAWINGS

[0009]FIG. 1 depicts the HBVPol sequence and deletion variant sequencesthereof.

[0010]FIG. 2 is a photograph showing in vitro binding assay for HBVPoland p11.

[0011]FIG. 3 is a photograph showing location of HBVPol and p11 in HepG2cells.

[0012]FIGS. 4a to 4 c are photographs showing function of p11 to deriveHBVPol to Promyclocytic Leukemia Nuclear Body (PMLNB) in HepG2 cells.

[0013]FIGS. 5a and 5 b are photographs showing that HBVPol/p11 complexmoves in a cell depending on concentration of Ca²⁺.

[0014]FIG. 6 is a photograph showing that if p11 is present in 2,2,1.5HepG2 cells in which HBV develops, the size or number of PMLNB is higherthan those of PMLNB in a normal cell.

[0015]FIG. 7 is a schematic diagram of plasmid pCMV-tag2A comprising asequence coding for HBVPol.

[0016]FIG. 8 is a schematic diagram of plasmid pCMV-tag3B comprising asequence coding for p11.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present disclosure is based on investigation of proteins in ahuman liver cell which bind to Hepatitis B virus polymerase (HBVPol)which is involved in Hepatitis B. It was found that Ca²⁺ binding proteinof 11 kDa forms a complex with HBVPol. Also, it was shown that theHBVPol/p11 complex moves to Promyelocytic Leukemia Nuclear Body (PMLNB)which is present in the nucleus of the studied cells and this phenomenonis performed by p11 under influence of intracellular calcium.

[0018] Thus, the present invention provides a complex of Ca²⁺ bindingprotein with HBVPol, wherein p11 binds to HBVPol in a non-covalentmanner.

[0019] The invention also encompasses a method for controlling movementof the complex into the nucleus of a cell depending on the intracellularconcentration of Ca²⁺ ion by administrating a controlling agent foradjusting the calcium ion concentration in a HepG2 cell. The controllingagent can increase or decrease the intracellular concentration of Ca²⁺ion.

[0020] A yeast two-hybrid system was used to search for a proteincapable of binding to HBVPol and to identify the location of both HBVPoland the protein in a human liver cell. A variety of yeast strains, suchas Saccharomyces cerevisiae strain AH109 and Y187 can be used in theyeast two-hybrid system, and all molecular biological manipulationexperiments with plasmids were carried out using E. coli strain DH5.These are provided by way of example and are not intended to limit thescope of microorganism strains which may be used.

[0021] The yeast two-hybrid system comprised a diploid of a yeastlibrary cell which was transformed with the genetic information of ahuman liver cell and a cell which had the HBVPol related gene. Yeastmating was carried out to screen for factors in the library cell whichbind to each HBVPol related proteins. The human Ca²⁺ binding protein(p11) of 11 kDa length was found to bind to HBVPol, and formed a complexwith HBVPol in the yeast two-hybrid system.

[0022] The p11 that was identified as binding to HBVPol was cloned in amammalian cell expression vector for application to a human liver celland then used to transform a human liver cell strain. The transformedhuman liver cell was reacted with an antibody against p11 and thenexamined by a confocal laser scanning microscope, with which thelocation of HBVPol and p11 in the liver cell was identified.

[0023] On the basis of the above, the transformed cells were stainedwith PML antibody and then observed under confocal laser scanningmicroscope. PMLNB and HBVPol were not in the same location in cellswherein only HBVPol is delivered. HBVPol migrates to PMLNB only in cellswherein p11 is delivered together with HBVPol. This indicates that p11plays an important role when HBVPol migrates to PMLNB in the human livercell.

[0024] It was also confirmed that when a cell co-delivered with HBVPoland p11 is treated with a calcium channel blocker, HBVPol and p11migrate to the nucleus of the cell. When cells co-delivered with HBVPoland p11 are treated with a calcium ion concentration increasing agent,HBVPol and p11 remain in the cytoplasm of the cell. Therefore, movementof the HBVPol/p11 complex in the cytoplasm to PMLNB in the nucleus canbe controlled by modulating calcium ion concentration in a cell.

[0025] The present invention will now be described in detail withreference to the following examples, which are not intended to limit thescope of the invention.

EXAMPLES

[0026] <Yeast and Bacteria Used in the Yeast Two-Hybrid System of theInvention>

[0027] Yeast used in the yeast two-hybrid system of the invention are asshown below and all molecular biological manipulation experiments ofplasmids were performed by using E. coli strain DH5.

[0028] Yeast Strain:

[0029]Saccharomyces cerevisiae AH109 (MATa, trp1-901, leu2-3, 112,ura3-52, his3-200, gal4, gal80, LYS2::GAL1UAS-GAL1TATA-HIS3,GAL2UAS-GAL2TATA-ADE 2URA3::MEL1UAS-MEL1TATA-lacZ, MEL1) (Clontech); and

[0030]Saccharomyces cerevisiae Y187 (MAT, ura3-52, his3-200, ade2-101,trp1-901, leu2-3, 112, gal4, gal80, met-, URA3::GAL1UAS-GAL1TATA lacZ,MEL1) (Clontech).

[0031] <Human Liver Cell Strain and Antibody>

[0032] HepG2 (ATCC number: HC-8065) was used as the human liver cellstrain. The antibodies used in the Western experiment,immunoprecipitation experiment and immuno fluorescent experiment wereanti-Myc mouse and rabbit antibody (Roche), anti-Flag, HA mouse andrabbit antibody (Santa Cruz Biotechnology) and anti-PML mouse antibody(Santa Cruz Biotechnology). To anti-rabbit goat antibody and antimousegoat secondary antibody were attached Rhodamine or FITC (fluoresceinisothiocyanate) (Santa Cruz Biotechnology).

Example 1

[0033] Yeast Two-Hybrid GAL4 System

[0034] Yeast (Y187) library (Clontech), which was transformed with thegenetic information of a human liver cell, was used to screen factors inthe cell, which bind to HBVPol, using MATCHMAKER yeast two-hybrid system(Clontech).

[0035] Ayw subtype HBVPol which was designed to have the restrictionsites of NdeI at 5′ end and SalI at 3′ end and 8 domain variants thereof(FIG. 1a) were reacted with 10 units of the restriction enzymes of NdeIand SalI for two hours. Thereafter, the reactants were amplified by PCRconsisting of 30 cycles of 95° C. 5 min, 95° C. 30 sec, 54° C. 1 min and72° C. 3 min and then 1 cycle of 72° C. 7 min. The amplified fragmentswere reacted with pGBKT7 (Clontech), which was digested with the samerestriction enzymes as above, in the presence of T4 DNA ligase of 4units at 16° C. for 20 hours to clone the amplified fragments in theplasmid.

[0036] The AH109 yeast which was intended to be transformed was platedon YPAD plate (1% yeast extract, 2% peptone, 2% dextrose (D-glucose),0.003% adenine, 2% agar) and grown at 30° C. for 24 hours. Thereafter,50 μl of yeast was placed into 1.5 ml tube with loop and was washed with1 ml of sterilized distilled water. The yeast pellet was dissolved in250 μl of plating solution (40% PEG3350, 0.01 M TE buffer (pH 7.5), 0.1M lithium acetate (pH 7.5)). To this solution, 5 μl of single strandedDNA (10 mg/ml), 10 μl of 1 M DTT and 1 μg of the above prepared plasmidpGBKT7 were added and stood at a room temperature for 6 hours. Heatshock was given to the cells at 42° C. for 15 minutes and thencentrifuged at 3,000 rpm for 1 minute. Thereafter, the supernatant wasdiscarded and the yeast pellet was washed with 1 ml of the sterilizeddistilled water. 100 μl of TE buffer (pH 7.5) was added to the yeastpellet. The resulting solution was plated on SD-Trp plate and the cellswere grown at 30° C.

[0037] The above yeast library cells containing the gene library of thehuman liver cell were grown in a liquid medium including no Leu(containing 0.67% yeast nitrogen base without amino acids, 20 mg/lL-arginine HCl, 30 mg/l L-isoleucine, 30 mg/l lysine HCl, 20 mg/lL-methionine, 50 mg/l L-phenylalanine, 200 mg/l L-threonine, 30 mg/lL-tyrosine, 20 mg/l L-uracil, 150 mg/l L-valine, 20 mg/l adenine, 20mg/l histidine, 20 mg/l tryptophan and 2% glucose) for 24 hours untilOD₆₀₀ reached 0.8.

[0038] At the same time, each of the yeast including HBVPol relatedgenes was grown in a liquid medium including no Trp (containing 0.67%yeast nitrogen base without amino acids, 20 mg/l L-arginine HCl, 30 mg/lL-isoleucine, 30 mg/l lysine HCl, 20 mg/l L-methionine, 50 mg/lL-phenylalanine, 200 mg/l L-threonine, 30 mg/l L-tyrosine, 20 mg/lL-uracil, 150 mg/l L-valine, 20 mg/l adenine, 20 mg/l histidine, 100mg/l leucine and 2% glucose) for 30 hours until OD₆₀₀ reached 0.9. Then,2 ml of the library cells and each of HBVPol of 50 ml were mixed with 45ml of YPDA yeast complex media and mated at 30° C. for 20 hours withstirring.

[0039] The resulting diploid yeasts were collected and plated on amedium plate including no leu, trp, ade and his (containing 0.67% yeastnitrogen base without amino acids, 20 mg/l L-arginine HCl, 30 mg/lL-isoleucine, 30 mg/l lysine HCl, 20 mg/l L-methionine, 50 mg/lL-phenylalanine, 200 mg/l L-threonine, 30 mg/l L-tyrosine, 20 mg/lL-uracil, 150 mg/l L-valine and 2% glucose) to select the diploid havingan interaction between HBVPol and the library proteins.

[0040] The genotypes of strain AH109 (MATa, trp1-901, leu2-3, 112,ura3-52, his3-200, gal4, gal80, LYS2::GAL1UAS-GAL1TATA-HIS3,GAL2UAS-GAL2TATA-ADE 2URA3:MEL1UAS-MEL1TATA-lacZ, MEL1) and Y187(MAT,ura3-52, his3-200, ade2-1, trp1-901, leu2-3, 112, gal4, gal80, met-,URA3::GAL1UAS-GAL1TATA LacZ, MEL1), the yeast diploid, wherein the genesincorporated in two yeasts are interacted, were able to grow on solidmedia including no Ade, His, Trp and Leu. This indicates that HBVPol ofAH109 interacts with the library proteins of Y187 (see, Bendixen, C.,Gangloff, S., Rothstein, R. (1994) A yeast mating-selection scheme fordetection of protein-protein interactions. Nucleic Acids Res., 22,1778-1779).

Example 2

[0041] Co-Immunoprecipitation Analysis

[0042] Ca²⁺ binding protein p11 of 11 kDa, which bind to HBVPol, wasscreened by using the yeast two-hybrid system and the binding wasconfirmed by using TNT coupled reticulocyte lysate System (Promega).

[0043] Since open reading frame coding for p11 is cloned to pGADT7 inY187 yeast cell (Clontech), p11 can be produced by bindingL-[35S]-methionine using T7 promoter. was reacted with 1 g of anti-HAmouse antibody at 4° C. for 2 hours. The reactants were precipitated byusing protein G plus/protein A agarose bead (Oncogene, Inc.) andanalyzed on 15% SDS-PAGE (FIG. 2, lane 1). HBVPol, which was cloned topGBKT7 as shown in Example 1, was also synthesized and then analyzed inthe same way as p11 (FIG. 2, lane 2).

[0044] The p11 and HBVPol were stood for 1 hour. Then, HBVPol wasreacted with 1 g of anti-Myc at 4° C. for 2 hours and p11 was reactedwith 1 g of HA mouse antibody at 4° C. for 2 hours. The reactants wereprecipitated by using protein G plus/protein A agarose bead (Oncogene,Inc.) and washed to remove any materials which did not bind to HBVPol orp11 having the antibodies bound thereto. Thereafter, the reactants werereacted with SDS loading buffer at 95° C. for 5 minutes and separatedfrom protein G plus/protein A agarose. The separated materials were runon 15% SDS-PAGE to analyze the radioactive products with X-ray film(FIG. 2, lanes 3 and 4).

[0045] Lane 3 of FIG. 2 is the result of experiment using anti-Myc mouseantibody for HBV Pol, lane 4 is the result of experiment using HA mouseantibody for p11. The lanes show that the bands of HBV Pol and p11 arepresent in a separate state, and this means that HBV Pol and p11 formthe complex by a non-covalent binding.

Example 3

[0046] Preparation of Plasmid for Expression of a Mammalian Cell

[0047] The p11 identified in Example 2 was cloned to a plasmid forexpression of a mammalian cell for applying the p11 to a human livercell.

[0048] HBV Pol was amplified by PCR consisting of 1 cycle of 95° C. 5minutes; 30 cycles of 95° C. 30 seconds, 54° C. 1 minute, 72° C. 3minutes; and 1 cycle of 72° C. 7 minutes, and then digested with 10units of HindIII and SalI for 2 hours. The restricted products wereligated to pCMV2A vector (Stratagene), which was restricted with theidentical restriction enzymes, with 4 units of ligase per reaction at16° C. for 20 hours to prepare pCMV-tag2A plasmid containing a HBVPolcoding sequence (FIG. 7).

[0049] The p11 was amplified by PCR consisting of 1 cycle of 95° C. 5minutes; 30 cycles of 95° C. 30 seconds, 55° C. 1 minute and 72° C. 1minutes; and 1 cycle of 72° C. 7 minutes, and then digested with 10units of EcoRI and XhoI for 2 hours. The restricted products wereligated to pCMV3B vector (Stratagene), which was restricted with theidentical restriction enzymes, with 4 units of ligase per reaction at16° C. for 20 hours to prepare pCMV-tag3B plasmid containing a p11coding sequence (FIG. 8).

Example 4

[0050] Immunofluorescent Experiment and Microscopy

[0051] HepG2 was grown on a chamber slide charged with DMEM(supplemented with 0.1 mM non-essential amino acids and 10% FBS,culturing condition: 37° C., moisturized with a mixture of 95:5% (v/v)air and CO₂), and Effectene (Qiagen) was used to deliver genes into thecell. Cells treated with EGTA and to which the gene was delivered, werewashed with PBS and fixed with 100% methanol at −20° C. for 15 minutes.The slide was treated with a blocking solution (1% goat serum/PBS) atroom temperature for 30 minutes and then reacted with anti-Myc rabbit(1:200) antibody against p11, anti-Flag rabbit (1:200) antibody againstHBVPol, or anti-PML mouse (1:200) antibody at 4° C. for one day.

[0052] After reaction, the cells were reacted with anti-rabbit oranti-mouse goat secondary antibody, to which rhodamine or fluorsceinisothiocyanate was attached, at 37° C. and then a coverslip was attachedthereto with mounting solution. Thereafter, the stained cells wereobserved with a confocal laser scanning microscope (CLSM Bio-Rad 1024).FIG. 3 shows photographs of HBVPol, p11 and overlap of two proteins fromthe left to the right and confirms that the proteins are present witheach other while forming dots in a cytoplasm, nucleus membrane andnucleus of HepG2 cell.

[0053] In view of the fact that HBVPol and p11 form dots in the nucleusof the cell, the cell was stained with PML antibody. As a result, it wasconfirmed that PMLNB was present in the same location with p11 andHBVPol as shown in FIG. 4a. Furthermore, in the cell to which onlyHBVPol was delivered, PMLNB was not present in the same location withHBVPol (FIG. 4b), while HBVPol moved to PMLNB only in the cell whereinp11 was delivered with HBVPol (FIG. 4c). From these observations, p11was confirmed to play an important role when HBVPol moves to PMLNB ofthe human liver cell.

[0054] Taking notice that p11 is a Ca²⁺ binding protein, a cell to whichHBVPol and p11 are co-delivered was treated with 0.5 mM of a Ca²⁺ ionconcentration decreasing agent, EGTA for 48 hours. As a result, it wasconfirmed that most of HBVPol and p11 migrated into the nucleus of thecell (FIG. 5a), while in cells treated with 30 uM of Ca²⁺ ionconcentration increasing agent, valinomycine for 24 hours, HBVPol andp11 remained in the cytoplasm of the cell (FIG. 5b).

[0055] As shown in the examples, it was confirmed that HBVPol binds top11 to form a complex and the complex moves into PMLNB in the nucleus ofthe cell, at which time, both p11 and a low concentration of Ca²⁺ ionare important factors for movement of HBVPol. Therefore, theintracellular location of p11 and HBVPol can be controlled by adjustingthe Ca²⁺ concentration in the cell.

[0056] Furthermore, 2,2,1.5 HepG2 was transformed with pCMV-tag3Bplasmid to only express p11. The p11 was reacted with anti-Myc rabbit(1:200) antibody against p11 and observed with a confocal laser scanningmicroscope (CLSM Bio-Rad 1024). When p11 is present in 2,2,1.5 HepG2cell wherein the HBV develops, the size and number of PMLNB wasconfirmed to be higher than those of PMLNB in a normal cell (FIG. 6).

[0057] Although it was reported that the size and number of PMLNB inhepatocellular carcinoma (HCC) patient by HBV infection increase as thecancer progresses (Yoon, G. S., 2001, J. Korean Med. Sci., 16, 433-438),it is the present invention that ascertains that increase of the size ornumber of PMLNB is caused by p11.

[0058] According to the present invention which confirms that p11induces movement of HBVPol to PMLNB of a liver cell depending on Ca²⁺concentration in the cell, various targets for treating liver cancer canbe provided.

What is claimed is:
 1. A complex of the 11 kDa Ca²⁺ binding protein p11and Hepatitis B virus polymerase (HBVPol), which moves to thePromyelocytic Leukemia Nuclear Body (PMLNB).
 2. A method for controllingmovement of the HBVPol/p11 complex into the nucleus of a HepG2 cellthrough adjustment of the intracellular concentration of Ca²⁺ ion byadministrating an agent which controls the calcium ion concentration inthe HepG2 cell.
 3. The method according to claim 2, wherein said agentwhich controls calcium ion concentration, increases said intracellularconcentration of Ca²⁺ ion.
 4. The method according to claim 2, whereinsaid agent which controls the calcium ion concentration, decreases saidintracellular concentration of Ca²⁺ ion.